JP7430944B2 - Method for producing acetylated sodium hyaluronate - Google Patents
Method for producing acetylated sodium hyaluronate Download PDFInfo
- Publication number
- JP7430944B2 JP7430944B2 JP2022560961A JP2022560961A JP7430944B2 JP 7430944 B2 JP7430944 B2 JP 7430944B2 JP 2022560961 A JP2022560961 A JP 2022560961A JP 2022560961 A JP2022560961 A JP 2022560961A JP 7430944 B2 JP7430944 B2 JP 7430944B2
- Authority
- JP
- Japan
- Prior art keywords
- reaction
- hyaluronate
- solution
- sodium hyaluronate
- producing
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
- C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
- C08B37/0072—Hyaluronic acid, i.e. HA or hyaluronan; Derivatives thereof, e.g. crosslinked hyaluronic acid (hylan) or hyaluronates
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
- C08L5/00—Compositions of polysaccharides or of their derivatives not provided for in groups C08L1/00 or C08L3/00
- C08L5/08—Chitin; Chondroitin sulfate; Hyaluronic acid; Derivatives thereof
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Organic Chemistry (AREA)
- Polymers & Plastics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Materials Engineering (AREA)
- Biochemistry (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
Description
本発明は有機化学合成の技術分野に関し、具体的には、アセチル化ヒアルロン酸ナトリウムの製造方法に関する。 The present invention relates to the technical field of organic chemical synthesis, and specifically relates to a method for producing acetylated sodium hyaluronate.
アセチル化ヒアルロン酸ナトリウムは、ヒアルロン酸ナトリウムをアセチル化反応させて得られる保湿原料である。ヒアルロン酸ナトリウムは水酸基上の水素がアセチルで部分的に置換されていることにより、親水性を維持しながら親油性を高め、肌との親和性や吸着性の向上に寄与する。アセチル化ヒアルロン酸ナトリウムは、みずみずしく、べたつかない手触りを有し、優れた保湿効果を発揮し、肌のバリアを修復し、肌のハリを改善し、肌をなめらかに保つことができる。アセチル化ヒアルロン酸ナトリウムの構造から見ると、二糖単位ごとに４つのヒドロキシ水素がアセチルに置換されるので、１つの二糖構造に対して置換度は０～４の範囲であり、置換度はヒアルロン酸ナトリウムの親水性、親油性に影響する。アセチル化改質の目的はヒアルロン酸ナトリウム親水性を維持しつつ親油性を向上させることであるため、ヒアルロン酸ナトリウムの親水性と親油性をバランスのとれた置換度にすることが好ましい。研究によると、アセチルの置換度は２．６～３．６の間であり、アセチル化ヒアルロン酸ナトリウムは親水性、親油性が最適である。 Acetylated sodium hyaluronate is a moisturizing raw material obtained by subjecting sodium hyaluronate to an acetylation reaction. In sodium hyaluronate, the hydrogen on the hydroxyl group is partially substituted with acetyl, which increases lipophilicity while maintaining hydrophilicity, contributing to improved affinity and adsorption with the skin. Acetylated sodium hyaluronate has a fresh, non-greasy feel, exhibits excellent moisturizing effects, can repair the skin barrier, improve skin firmness, and keep skin smooth. Looking at the structure of acetylated sodium hyaluronate , four hydroxy hydrogens are substituted with acetyl in each disaccharide unit, so the degree of substitution ranges from 0 to 4 for one disaccharide structure, and the degree of substitution is Affects the hydrophilicity and lipophilicity of sodium hyaluronate . Since the purpose of the acetylation modification is to improve the lipophilicity of sodium hyaluronate while maintaining its hydrophilicity, it is preferable to adjust the degree of substitution so that the hydrophilicity and lipophilicity of sodium hyaluronate are well-balanced. According to research, the degree of acetyl substitution is between 2.6 and 3.6, and acetylated sodium hyaluronate is optimally hydrophilic and lipophilic.
従来技術のアセチル化ヒアルロン酸ナトリウムの製造方法では、酢酸／無水酢酸を溶媒とし、濃硫酸で触媒することによりアセチル化ヒアルロン酸ナトリウムが得られるが、濃硫酸を触媒とするので、危険性が高く、その後エタノールで沈殿させる必要があり、アセチル化ヒアルロン酸ナトリウムは通常のヒアルロン酸ナトリウムに比べてエタノールへの溶解性が高いため、より多くの無水エタノールで沈殿させる必要があり、多大なコストがかかる。 In the conventional method for producing acetylated sodium hyaluronate, acetylated sodium hyaluronate can be obtained by using acetic acid/acetic anhydride as a solvent and catalyzing with concentrated sulfuric acid, but since concentrated sulfuric acid is used as a catalyst, it is highly dangerous. , then it needs to be precipitated with ethanol, and acetylated sodium hyaluronate has a higher solubility in ethanol than normal sodium hyaluronate, so it needs to be precipitated with more absolute ethanol, which incurs great cost. .
出願番号２０１９１１０１５４８１２の中国発明特許出願に開示されたアセチル化ヒアルロン酸ナトリウムの製造方法のように、ＤＭＦを溶媒とし、アセチル化クロライドをアシル化剤とする方法もあり、保護ガス下、ヒアルロン酸ナトリウム塩とアセチル化クロライドとを有機溶媒にて反応させ、生成物を分離して精製した後、アルカリと塩化して製品を得る。このプロセスは、濃硫酸などの腐食性やピリジン類の精製困難な触媒を必要としないが、アセチルクロライドは腐食性が強く、生産中に設備を損耗しやすく、また製品の鉄、クロムの含有量が基準値を超えることを引き起こしやすく、プロセスの後処理においてｐＨを１２まで調整する必要があり、アルカリ性環境下でエステル基が加水分解しやすいため、アセチル置換度が低下し、過酸化物の脱色も安全上のリスクが存在する。 There is also a method for producing acetylated sodium hyaluronate using DMF as a solvent and acetylated chloride as an acylating agent, such as the method for producing acetylated sodium hyaluronate disclosed in the Chinese invention patent application with application number 2019110154812. and acetylated chloride are reacted in an organic solvent, the product is separated and purified, and then salted with an alkali to obtain a product. This process does not require corrosive catalysts such as concentrated sulfuric acid or difficult-to-purify catalysts such as pyridines, but acetyl chloride is highly corrosive and easily wears out equipment during production. It is easy to cause the pH to exceed the standard value, it is necessary to adjust the pH to 12 in post-processing, and the ester group is easy to hydrolyze in an alkaline environment, which reduces the degree of acetyl substitution and decolorizes the peroxide. There are also safety risks.
本発明が解決しようとする技術的課題は、従来技術の欠点に対して、置換度の過不足を効果的に回避し、アセチル置換度が２．７～３．２の生成物ができ、反応原料及び条件が温和で、分離精製のステップが簡単であるアセチル化ヒアルロン酸ナトリウムの製造方法を提供することである。 The technical problem to be solved by the present invention is to effectively avoid excess or deficiency in the degree of substitution, to produce a product with a degree of acetyl substitution of 2.7 to 3.2, and to react with the disadvantages of the prior art. It is an object of the present invention to provide a method for producing acetylated sodium hyaluronate using mild raw materials and mild conditions and simple separation and purification steps.
本発明が上記の技術的課題を解決する技術的解決手段は以下の通りである。アセチル化ヒアルロン酸ナトリウムの製造方法であって、不活性ガスの保護条件下、ヒアルロン酸塩と無水酢酸を有機溶剤中にて、触媒で反応を触発し、反応中に吸水剤を加え、反応液を後処理して、分離して精製し、乾燥して生成物を得るステップを含むことを特徴とし、得た生成物をヒドロキシルアミン比色法で検出した結果、アセチル置換度は２．７～３．２である。 The technical solutions by which the present invention solves the above technical problems are as follows. A method for producing acetylated sodium hyaluronate, which involves catalyzing the reaction of hyaluronate and acetic anhydride with a catalyst in an organic solvent under the protection of an inert gas, adding a water-absorbing agent during the reaction, and dissolving the reaction solution. The product is characterized by including a step of post-processing, separating, purifying, and drying to obtain a product, and as a result of detecting the obtained product by a hydroxylamine colorimetric method, the degree of acetyl substitution is 2.7 ~ It is 3.2.
さらに、ヒアルロン酸塩と無水酢酸の反応においては、ヒアルロン酸塩を均一相となるまで有機溶剤に溶解し、吸水剤を加え、反応系を－５℃～１０℃に降温し、反応系に無水酢酸を滴下し、次に反応系にＤＭＡＰ－トルエン溶液を滴下し、所定時間反応させた後、４０～７０℃に加熱して所定時間反応させ、淡黄色の混濁溶液を得る。 Furthermore, in the reaction between hyaluronate and acetic anhydride, the hyaluronate is dissolved in an organic solvent until it becomes a homogeneous phase, a water-absorbing agent is added, the temperature of the reaction system is lowered to -5°C to 10°C, and anhydrous is added to the reaction system . Acetic acid is added dropwise, and then a DMAP-toluene solution is added dropwise to the reaction system , and the mixture is allowed to react for a predetermined time, and then heated to 40 to 70°C and allowed to react for a predetermined time to obtain a pale yellow cloudy solution.
無水酢酸を滴下するときに降温して滴下するのは、無水酢酸の濃度が局所的に高すぎて反応が速く、局所放熱により置換度が高すぎるフロック沈殿が生成されることを防止するためである。一般には、激しい撹拌及び降温滴下により対応することができるが、磁気撹拌や小型機械による撹拌の場合は効果が不十分であるので、降温後滴下し、系を均一に撹拌すると、常温に自然回復して反応させる。 The reason why the temperature is lowered when dropping acetic anhydride is to prevent the concentration of acetic anhydride from being locally too high and the reaction to be rapid, resulting in the formation of floc precipitates with too high a degree of substitution due to local heat dissipation. be. In general, this can be achieved by vigorous stirring and dropping the temperature while cooling, but magnetic stirring or stirring using a small machine is not sufficiently effective, so if you drop the mixture after cooling and stir the system uniformly, the temperature will naturally return to room temperature. and react.
反応式は以下の通りである。
反応中に吸水剤を加えることは、多糖鎖に含まれる水分をできる限り吸収し、水が先に無水酢酸と反応して無水酢酸の一部を消費することによる置換度低下を防止することを目的とする。 Adding a water absorbing agent during the reaction absorbs as much water contained in the polysaccharide chain as possible, and prevents a decrease in the degree of substitution caused by water reacting with acetic anhydride first and consuming a portion of acetic anhydride. purpose.
さらに、前記ヒアルロン酸塩と無水酢酸との質量比が１：１～１：５であり、ヒアルロン酸塩の平均分子量が５ＫＤａ～５０００ＫＤａ、好ましくは１０ＫＤａ～２０００ＫＤａ、より好ましくは２０ＫＤａ～２００ＫＤａである。 Further, the mass ratio of the hyaluronate to acetic anhydride is 1:1 to 1:5, and the average molecular weight of the hyaluronate is 5KDa to 5000KDa, preferably 10KDa to 2000KDa, and more preferably 20KDa to 200KDa.
さらに、触媒反応終了後、反応液の後処理過程は、反応液に水を加えて、残りの無水酢酸をクエンチし、吸水剤をろ過除去することである。 Moreover, after the catalytic reaction is completed, the post-treatment process of the reaction solution is to add water to the reaction solution to quench the remaining acetic anhydride, and to filter out the water-absorbing agent.
さらに、前記吸水剤は、撹拌破砕可能であるが、反応に関与することがなく、そしてワンステップろ過により除去できる分子篩を使用し、前記触媒はＤＭＡＰを使用し、ヒアルロン酸塩とＤＭＡＰとの質量比が１：０．０５～１：０．１２である。 Further, the water absorbing agent uses a molecular sieve that can be crushed by stirring but does not participate in the reaction and can be removed by one-step filtration, the catalyst uses DMAP, and the mass of hyaluronate and DMAP is The ratio is 1:0.05 to 1:0.12.
さらに、前記有機溶剤はトルエンを用い、材料溶媒比が１：５～１：２０である。 Furthermore, toluene is used as the organic solvent, and the material-solvent ratio is 1:5 to 1:20.
さらに、反応液の分離精製過程は、反応液を膜でろ過して精製することであり、精製中に、留分が無色かつ弱酸性となるまで、水を少量ずつ複数回加える。使用される膜は、トルエンなどの有機溶剤に強く、膜孔径が２００ＫＤ～５０００ＫＤである有機膜又はセラミックス膜を使用する。 Furthermore, the separation and purification process of the reaction solution involves purifying the reaction solution by filtering it through a membrane, and during the purification, water is added in small portions multiple times until the fraction becomes colorless and weakly acidic. The membrane used is an organic membrane or a ceramic membrane that is resistant to organic solvents such as toluene and has a membrane pore diameter of 200 KD to 5000 KD.
さらに、分離精製後、反応液をアルカリ液でｐＨ５～７に調整し、濃縮する。ここで、アルカリ液は水酸化ナトリウム溶液、炭酸ナトリウム溶液、重炭酸ナトリウム溶液のうちの少なくとも１種を使用する。 Furthermore, after separation and purification, the reaction solution is adjusted to pH 5 to 7 with an alkaline solution and concentrated. Here, as the alkaline solution, at least one of a sodium hydroxide solution, a sodium carbonate solution, and a sodium bicarbonate solution is used.
さらに、前記乾燥過程は、温度０～２５℃の低温乾燥を採用する。 Furthermore, the drying process employs low-temperature drying at a temperature of 0 to 25°C.
本発明の有益な効果は以下の通りである。本発明のプロセスでは、ヒアルロン酸塩と無水酢酸の反応中に吸水剤及び分子篩を加え、吸水剤で水分による反応への干渉を解消し、水が先に無水酢酸と反応して無水酢酸の一部を消費することによる置換度低下を防止し、また、反応温度を制御することにより、置換度の過不足の問題を解消する。膜でろ過精製し、この過程において水を少量ずつ複数回加えることで、導入される触媒や有機溶剤を除去する。大量のエタノールで沈殿させることを省略し、消耗を低下させ、しかも、得た生成物は高純度で、形態が良好である。プロセスは全体として反応条件が温和であり、分離精製のステップが簡単である。 The beneficial effects of the present invention are as follows. In the process of the present invention, a water-absorbing agent and a molecular sieve are added during the reaction of hyaluronate and acetic anhydride, and the water-absorbing agent eliminates the interference of water with the reaction, so that the water reacts with acetic anhydride first, resulting in the reaction of acetic anhydride. By preventing a decrease in the degree of substitution due to consumption of parts, and by controlling the reaction temperature, the problem of excess or deficiency in the degree of substitution can be solved. It is purified by filtration through a membrane, and by adding water in small quantities multiple times during this process, the introduced catalyst and organic solvent are removed. Precipitation with a large amount of ethanol is omitted, reducing consumption, and the obtained product is of high purity and good morphology. The overall reaction conditions of the process are mild, and the separation and purification steps are simple.
以下、本発明の原理及び特徴が説明されるが、記載される実施例は本発明を解釈するために過ぎず、本発明の範囲を限定するものではない。 Hereinafter, the principles and features of the present invention will be explained, but the described examples are only for interpreting the present invention and are not intended to limit the scope of the present invention.
実施例１
本実施例のアセチル化ヒアルロン酸ナトリウムの製造方法は、以下のステップを含む。
（１）窒素ガスの保護下、１０００ｍＬ反応フラスコに分子量１０ＫＤａのヒアルロン酸ナトリウムを５０．００ｇ秤量し、無水溶媒であるトルエン２５０ｍＬに加えて６０℃に昇温して撹拌し、全てが溶解して均一相となると、反応中に吸水するための分子篩を加え、次に、反応系を－５℃に降温し、反応液に無水酢酸５０ｍＬを滴下し、次に、ＤＭＡＰ ０．２５ｇをトルエン溶液４ｍＬで溶解したものを反応液に滴下し、常温で１ｈ反応させた後、４０℃に加熱して４ｈ反応させ、淡黄色の混濁溶液を得る。
（２）等体積の水を加えて、残りの無水酢酸をクエンチし、反応液をろ過して分子篩を除去する。
（３）反応液を膜でろ過して精製し、精製中に、留分が無色かつ弱酸性となるまで水を少量ずつ複数回加え、最後の精製が完了すると、反応液の体積は２００ｍＬとなる。
（４）反応液を水酸化ナトリウム溶液でｐＨ５～７に調整し、濃縮する。
（５）乾燥してアセチル化ヒアルロン酸ナトリウム３９．０１ｇを得て、収率は６０％である。
得た生成物は結晶物形態であり、ヒドロキシルアミン比色法により検出したところ、アセチル置換度は２．９であり、２．６～３．６という規定範囲にある。
ここで、収率μ＝４０１．３＊ｍ2／ｍ1＊（４０１．３＋（４３－１）＊ＤＳ）
ｍ1はヒアルロン酸ナトリウム投入質量、ｍ2は得たアセチル化ヒアルロン酸ナトリウムの質量、ＤＳはアセチル置換度である。
Example 1
The method for producing acetylated sodium hyaluronate of this example includes the following steps.
(1) Under the protection of nitrogen gas, 50.00 g of sodium hyaluronate with a molecular weight of 10 KDa was weighed into a 1000 mL reaction flask, added to 250 mL of toluene, an anhydrous solvent, and heated to 60°C and stirred until everything was dissolved. When a homogeneous phase is obtained, molecular sieves are added to absorb water during the reaction, the temperature of the reaction system is lowered to -5°C, 50 mL of acetic anhydride is added dropwise to the reaction solution, and 0.25 g of DMAP is added to 4 mL of a toluene solution. The solution dissolved in above was added dropwise to the reaction solution, and the mixture was reacted at room temperature for 1 hour, then heated to 40° C. and reacted for 4 hours to obtain a pale yellow turbid solution.
(2) Add an equal volume of water to quench the remaining acetic anhydride, and filter the reaction solution to remove the molecular sieve.
(3) Purify the reaction solution by filtering it through a membrane, and during purification, add water several times in small amounts until the fraction becomes colorless and weakly acidic. When the final purification is completed, the volume of the reaction solution is 200 mL. Become.
(4) Adjust the reaction solution to pH 5-7 with sodium hydroxide solution and concentrate.
(5) After drying, 39.01 g of acetylated sodium hyaluronate was obtained, with a yield of 60%.
The product obtained is in crystalline form and the degree of acetyl substitution is 2.9, as detected by hydroxylamine colorimetry, in the specified range of 2.6 to 3.6.
Here, yield μ=401.3*m 2 /m 1 *(401.3+(43-1)*DS)
m 1 is the input mass of sodium hyaluronate , m 2 is the mass of the obtained acetylated sodium hyaluronate , and DS is the degree of acetyl substitution.
実施例２
本実施例のアセチル化ヒアルロン酸ナトリウムの製造方法は、以下のステップを含む。
（１）窒素ガス保護下、１０００ｍＬ反応フラスコに分子量１０００ＫＤａのヒアルロン酸ナトリウムを３０．００ｇ秤量し、無水溶媒であるトルエン３００ｍＬに加えて７０℃に昇温して撹拌し（昇温はヒアルロン酸ナトリウムを溶解することが目的であり、分子量が大きいほど、溶解が困難である）、全てが溶解して均一相となると、反応中に吸水するための分子篩を加え、次に、反応系を０℃に降温し、反応液に無水酢酸９０ｍＬを滴下し、次に、ＤＭＡＰ ０．２ｇをトルエン４ｍＬで溶解したものを反応液に滴下し、常温で１ｈ反応させた後、５０℃に加熱して１２ｈ反応させ、淡黄色の混濁溶液を得る。
（２）等体積の水を加えて、残りの無水酢酸をクエンチし、反応液をろ過して分子篩を除去する。
（３）反応液を膜でろ過して精製し、精製中に、留分が無色かつ弱酸性となるまで水を少量ずつ複数回加え、最後の精製が完了すると、反応液体積は約５００ｍＬとなる。
（４）反応液を炭酸ナトリウム溶液でｐＨ５～７に調整し、濃縮する。
（５）乾燥してアセチル化ヒアルロン酸ナトリウム１７．５ｇを得て、収率は５８％である。
得た生成物は結晶形態であり、ヒドロキシルアミン比色法により検出したところ、アセチル置換度は２．７であり、２．６～３．６の規定範囲である。
Example 2
The method for producing acetylated sodium hyaluronate of this example includes the following steps.
(1) Under nitrogen gas protection, 30.00 g of sodium hyaluronate with a molecular weight of 1000 KDa was weighed into a 1000 mL reaction flask, added to 300 mL of toluene, an anhydrous solvent, heated to 70°C, and stirred (the temperature was raised to 70°C). (The larger the molecular weight, the more difficult it is to dissolve.) Once everything has dissolved into a homogeneous phase, a molecular sieve is added to absorb water during the reaction, and the reaction system is then heated to 0°C. 90 mL of acetic anhydride was added dropwise to the reaction solution, and then a solution of 0.2 g of DMAP dissolved in 4 mL of toluene was added dropwise to the reaction solution. After reacting at room temperature for 1 hour, it was heated to 50 °C for 12 hours. The reaction yields a pale yellow cloudy solution.
(2) Add an equal volume of water to quench the remaining acetic anhydride, and filter the reaction solution to remove the molecular sieve.
(3) Purify the reaction solution by filtering it through a membrane, and during the purification, add water in small portions multiple times until the fraction becomes colorless and weakly acidic. When the final purification is completed, the reaction liquid volume will be approximately 500 mL. Become.
(4) Adjust the reaction solution to pH 5-7 with sodium carbonate solution and concentrate.
(5) After drying, 17.5 g of acetylated sodium hyaluronate was obtained, with a yield of 58%.
The product obtained is in crystalline form and the degree of acetyl substitution is 2.7, as detected by hydroxylamine colorimetry, in the specified range from 2.6 to 3.6.
実施例３
本実施例のアセチル化ヒアルロン酸ナトリウムの製造方法は、以下のステップを含む。
（１）窒素ガスの保護下、１０００ｍＬ反応フラスコに分子量２００ＫＤａのヒアルロン酸ナトリウムを２０．００ｇ秤量し、無水溶媒であるトルエン４００ｍＬに加えて７０℃に昇温して撹拌し、全てが溶解して均一相となると、反応中に吸水するための分子篩を加え、次に、反応系を－５℃に降温し、反応液に無水酢酸１００ｍＬを低下し、次に、ＤＭＡＰ ０．２４ｇをトルエン４ｍＬで溶解したものを反応液に滴下し、常温で１ｈ反応させた後、７０℃に加熱して４８ｈ反応させ、淡黄色の混濁溶液を得る。
（２）等体積の水を加えて、残りの無水酢酸をクエンチし、反応液をろ過して分子篩を除去する。
（３）反応液を膜でろ過して精製し、精製中に、留分が無色かつ弱酸性となるまで水を少量ずつ複数回加え、最後の精製が完了すると、反応液体積は１０００ｍＬとなる。
（４）反応液を重炭酸ナトリウム溶液でｐＨ５～７に調整し、濃縮する。
（５）乾燥してアセチル化ヒアルロン酸ナトリウム１３．８ｇを得て、収率は約５２％である。
得た生成物は結晶物形態であり、ヒドロキシルアミン比色法により検出したところ、アセチル置換度は３．１７であり、２．６～３．６の規定範囲である。
Example 3
The method for producing acetylated sodium hyaluronate of this example includes the following steps.
(1) Under the protection of nitrogen gas, 20.00 g of sodium hyaluronate with a molecular weight of 200 KDa was weighed into a 1000 mL reaction flask, added to 400 mL of toluene, an anhydrous solvent, and heated to 70°C and stirred until everything was dissolved. When a homogeneous phase is obtained, molecular sieves are added to absorb water during the reaction, the temperature of the reaction system is lowered to -5°C, 100 mL of acetic anhydride is added to the reaction solution, and 0.24 g of DMAP is added with 4 mL of toluene. The dissolved substance was added dropwise to the reaction solution and reacted at room temperature for 1 hour, then heated to 70° C. and reacted for 48 hours to obtain a pale yellow cloudy solution.
(2) Add an equal volume of water to quench the remaining acetic anhydride, and filter the reaction solution to remove the molecular sieve.
(3) Purify the reaction liquid by filtering it through a membrane, and during purification, add water in small quantities multiple times until the fraction becomes colorless and weakly acidic. When the final purification is completed, the reaction liquid volume will be 1000 mL. .
(4) Adjust the reaction solution to pH 5-7 with sodium bicarbonate solution and concentrate.
(5) After drying, 13.8 g of acetylated sodium hyaluronate was obtained, with a yield of about 52%.
The product obtained is in crystalline form and the degree of acetyl substitution is 3.17, as detected by hydroxylamine colorimetry, in the specified range of 2.6-3.6.
比較例１
製造方法は、方法においては吸水剤が添加されず、得たアセチル化ヒアルロン酸ナトリウムの置換度は２．２１である以外、実施例１と同様であった。その結果、この反応においてもアシル化反応が起こったが、置換度が低い。その原因としては、ヒアルロン酸ナトリウムは高い保水能力を持ち、構造を破壊せずにその中の水分を完全に除去することが困難であり、一般的な化粧品レベルのヒアルロン酸ナトリウムの乾燥重量減少が６％～９％の間であり、ヒアルロン酸ナトリウムによる反応系の水が無水酢酸と加水分解反応を行い、酢酸を生成し、無水酢酸の一部を消費し、このアシル化反応はヒドロキシ水素をアシル化して置換し、可逆的なエステル化反応でもあり、系には水が含まれるので、後続の逆反応が促進され、置換度が影響を受ける。
Comparative example 1
The manufacturing method was similar to Example 1, except that no water absorbing agent was added in the method and the degree of substitution of the acetylated sodium hyaluronate obtained was 2.21. As a result, although an acylation reaction occurred in this reaction as well, the degree of substitution was low. The reason for this is that sodium hyaluronate has a high water-holding capacity, and it is difficult to completely remove the water within it without destroying its structure. The water in the reaction system with sodium hyaluronate undergoes a hydrolysis reaction with acetic anhydride to produce acetic acid and consume a portion of acetic anhydride, and this acylation reaction converts hydroxy hydrogen into It is an acylation and substitution, and is also a reversible esterification reaction, and since water is included in the system, the subsequent reverse reaction is promoted and the degree of substitution is affected.
比較例２
製造方法は、吸水剤である分子篩を濃硫酸に変更し、その結果、吸水剤は系の酸アルカリ度に大きな影響を与え、さらに反応に直接関与する以外、実施例１とほぼ同様であった。
Comparative example 2
The production method was almost the same as in Example 1, except that the molecular sieve, which is a water absorbing agent, was changed to concentrated sulfuric acid, and as a result, the water absorbing agent had a large effect on the acid alkalinity of the system, and was also directly involved in the reaction. .
比較例３
製造方法は、吸水剤である分子篩をソーダライムに変更し、その結果、吸水剤は系の酸アルカリ度に大きな影響を与え、さらに反応に直接関与する以外、実施例１とほぼ同様であった。
Comparative example 3
The production method was almost the same as in Example 1, except that the molecular sieve, which is a water absorbing agent, was changed to soda lime, and as a result, the water absorbing agent had a large effect on the acid alkalinity of the system, and was also directly involved in the reaction. .
比較例４
製造方法は、吸水剤である分子篩を無水硫酸ナトリウムに変更し、実験の結果、無水硫酸ナトリウムは吸水後に直接ケーキングし、撹拌に悪影響を与える以外、実施例１とほぼ同じであった。
Comparative example 4
The manufacturing method was almost the same as in Example 1 except that the molecular sieve, which is a water absorbing agent, was changed to anhydrous sodium sulfate, and as a result of the experiment, anhydrous sodium sulfate caused direct caking after water absorption, which adversely affected stirring.
比較例５
製造方法は、ステップ（１）の温度が常温に制御され、残りの条件が同様である以外、実施例１とほぼ同じであった。その結果、ヒアルロン酸ナトリウムを溶解するのに時間がかかり、無水酢酸を滴下するときに降温しないので、滴下したところにフロック状物が現れ、次に、反応系の局所から放熱が起こり、滴下したところのフロック状物が多くなる。このようなフロック状物を採取して検出したところ、このフロック状物は、置換度が約４と高いアセチル化ヒアルロン酸ナトリウムであり、水に不溶であり、親水性を失い、不合格のものである。
Comparative example 5
The manufacturing method was almost the same as Example 1 except that the temperature in step (1) was controlled to room temperature and the remaining conditions were the same. As a result, it takes time to dissolve sodium hyaluronate, and the temperature does not drop when acetic anhydride is added dropwise, so flocs appear at the point where the acetic anhydride is dropped, and then heat is dissipated locally from the reaction system, causing the drop to drop. However, the amount of floc-like substances increases. When such floc-like substances were collected and detected, they were found to be acetylated sodium hyaluronate with a high degree of substitution of approximately 4, and were insoluble in water, losing hydrophilicity and being rejected. It is.
Claims (9)
前記吸水剤は分子篩を用い、前記触媒はＤＭＡＰを用い、
前記ヒアルロン酸塩と無水酢酸との反応においては、ヒアルロン酸塩を均一相となるまで前記有機溶剤に溶解し、前記吸水剤を加え、反応系を－５℃～１０℃に降温し、前記反応系に無水酢酸を滴下し、次に前記反応系にＤＭＡＰ－トルエン溶液を滴下し、所定時間反応させた後、４０～７０℃に加熱して所定時間反応させることを特徴とするアセチル化ヒアルロン酸ナトリウムの製造方法。 Under inert gas protection, hyaluronate and acetic anhydride are placed in an organic solvent to trigger a reaction with a catalyst, a water absorbing agent is added during the reaction, the reaction solution is post-treated, separated, purified, and dried. to obtain a product;
The water absorbing agent uses a molecular sieve, the catalyst uses DMAP,
In the reaction between the hyaluronate and acetic anhydride, the hyaluronate is dissolved in the organic solvent until it becomes a homogeneous phase, the water absorbing agent is added, the reaction system is cooled to -5°C to 10°C, and the reaction is carried out. Acetylated hyaluronic acid characterized by dropping acetic anhydride into the system, then dropping a DMAP-toluene solution into the reaction system, reacting for a predetermined time, and then heating to 40 to 70°C and reacting for a predetermined time. Method for producing sodium.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110427110.6 | 2021-04-21 | ||
| CN202110427110.6A CN113121721B (en) | 2021-04-21 | 2021-04-21 | Preparation method of acetylated sodium hyaluronate |
| PCT/CN2022/078460 WO2022222607A1 (en) | 2021-04-21 | 2022-02-28 | Preparation method for acetylated sodium hyaluronate |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JP2023525650A JP2023525650A (en) | 2023-06-19 |
| JP7430944B2 true JP7430944B2 (en) | 2024-02-14 |
Family
ID=76778495
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2022560961A Active JP7430944B2 (en) | 2021-04-21 | 2022-02-28 | Method for producing acetylated sodium hyaluronate |
Country Status (5)
| Country | Link |
|---|---|
| US (1) | US12435162B2 (en) |
| EP (1) | EP4119586B1 (en) |
| JP (1) | JP7430944B2 (en) |
| CN (1) | CN113121721B (en) |
| WO (1) | WO2022222607A1 (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113121721B (en) | 2021-04-21 | 2022-02-15 | 山东焦点福瑞达生物股份有限公司 | Preparation method of acetylated sodium hyaluronate |
| CN115558688A (en) * | 2022-10-20 | 2023-01-03 | 成都格纯生物医药有限公司 | A kind of fully acetylated sodium hyaluronate and preparation method thereof |
| CN120040619B (en) * | 2025-03-12 | 2025-09-30 | 山东众山生物科技有限公司 | Preparation method of acetylated sodium hyaluronate |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109206537A (en) | 2018-10-10 | 2019-01-15 | 华熙福瑞达生物医药有限公司 | A kind of preparation method and applications of acetylation Sodium Hyaluronate |
Family Cites Families (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US2170002A (en) * | 1935-05-18 | 1939-08-22 | Shawinigan Chem Ltd | Process of making acetic anhydride |
| US4288592A (en) * | 1979-04-30 | 1981-09-08 | American Cyanamid Company | Process for preparing amides by reaction in presence of molecular sieve |
| JP3142415B2 (en) * | 1992-04-21 | 2001-03-07 | 株式会社資生堂 | High acetylation rate hyaluronic acid and method for producing the same |
| DE69524197T2 (en) * | 1994-08-11 | 2002-08-14 | Shiseido Co. Ltd., Tokio/Tokyo | LOW MOLECULAR WEIGHT ACETYLATED HYALURONIC ACID; SOFTENERS AND METHOD FOR PRODUCING AND PURIFYING THIS ACID |
| JP3556975B2 (en) * | 1994-08-11 | 2004-08-25 | 株式会社資生堂 | Production method and purification method of acetylated hyaluronic acid |
| CN101171265B (en) * | 2005-05-04 | 2012-02-01 | 诺维信生物制药丹麦公司 | Method of controlling the content of selected component(s) from polymer(s) using molecular sieve(s) |
| EP1942117A1 (en) * | 2006-12-29 | 2008-07-09 | Sigea S.R.L. | Derivatives of acid polysaccharides |
| CN109232770A (en) * | 2018-09-07 | 2019-01-18 | 山东焦点生物科技股份有限公司 | A kind of preparation method of high substituted degree acetylation Sodium Hyaluronate |
| CN110467691A (en) | 2019-09-23 | 2019-11-19 | 山东银河生物科技有限公司 | A method of preparing acetylation hyaluronic acid |
| CN110724171A (en) * | 2019-10-24 | 2020-01-24 | 山东众山生物科技有限公司 | Preparation method of acetylated sodium hyaluronate |
| CN110981991B (en) * | 2019-12-24 | 2021-12-07 | 江苏诚信药业有限公司 | Preparation method of acetylated hyaluronate |
| CN113121721B (en) | 2021-04-21 | 2022-02-15 | 山东焦点福瑞达生物股份有限公司 | Preparation method of acetylated sodium hyaluronate |
-
2021
- 2021-04-21 CN CN202110427110.6A patent/CN113121721B/en active Active
-
2022
- 2022-02-28 US US17/918,906 patent/US12435162B2/en active Active
- 2022-02-28 WO PCT/CN2022/078460 patent/WO2022222607A1/en not_active Ceased
- 2022-02-28 EP EP22782444.8A patent/EP4119586B1/en active Active
- 2022-02-28 JP JP2022560961A patent/JP7430944B2/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109206537A (en) | 2018-10-10 | 2019-01-15 | 华熙福瑞达生物医药有限公司 | A kind of preparation method and applications of acetylation Sodium Hyaluronate |
Also Published As
| Publication number | Publication date |
|---|---|
| US20230295351A1 (en) | 2023-09-21 |
| EP4119586A4 (en) | 2023-11-08 |
| CN113121721B (en) | 2022-02-15 |
| EP4119586C0 (en) | 2025-02-12 |
| EP4119586B1 (en) | 2025-02-12 |
| WO2022222607A1 (en) | 2022-10-27 |
| EP4119586A1 (en) | 2023-01-18 |
| US12435162B2 (en) | 2025-10-07 |
| JP2023525650A (en) | 2023-06-19 |
| CN113121721A (en) | 2021-07-16 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP7430944B2 (en) | Method for producing acetylated sodium hyaluronate | |
| CN114736316B (en) | Green preparation method and application of acetylated hyaluronate | |
| CN110724171A (en) | Preparation method of acetylated sodium hyaluronate | |
| CN119019268A (en) | A method for preparing high-purity olaflurane raw material | |
| US20120264929A1 (en) | Method for producing n-acetyl-d-glucosamine | |
| CN1814609B (en) | A method for improving the synthesis yield of sucralose | |
| WO2011023027A1 (en) | Method for preparing prasugrel | |
| WO2022141228A1 (en) | Preparation method for sucralose, crude product solution and sucralose | |
| HUP0302530A2 (en) | Process for the purification of 2-nitro-4-methylsulphonylbenzoic acid | |
| CN102093444A (en) | Method for preparing isepamicin and salts thereof | |
| CN113694878A (en) | Preparation method and application of magnesium silicate adsorbent | |
| US10889654B2 (en) | Acylation process | |
| CN112409420B (en) | Purification method of 2 '-fluoro-2' -deoxyuridine | |
| CN106478722A (en) | High-purity L ascorbic acid 2 magnesium phosphorate salt production process | |
| CN110183432B (en) | Preparation process of 5, 6-O-isopropylidene-L-ascorbic acid | |
| CN112574258A (en) | Method for controlling sucralose acidity | |
| SU576937A3 (en) | Method of preparing mono-o-b-oxoethyl-7-rutine | |
| CN112694488B (en) | Synthesis method of L-type cefamandole nafate | |
| CN110642722A (en) | Method for preparing N, N-tetramethyl decamethylene diamine | |
| CN117343111B (en) | Preparation method of nucleoside modification N2-isobutyryl-2'-methoxyguanosine | |
| JPH0873399A (en) | Anhydrous trimagnesium citrate and its production | |
| CN110590591A (en) | Preparation method of iodixanol and iohexol impurities | |
| CN118184529A (en) | Preparation method of low-chloride mesalazine | |
| CN120136825A (en) | A method for synthesizing L-ascorbyl palmitate by acyl chloride method | |
| TW200526564A (en) | Method for producing 2,3,6,7,10,11-hexahydroxytriphenylene |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20221004 |
|
| A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20221004 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20230926 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20231102 |
|
| TRDD | Decision of grant or rejection written | ||
| A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20240116 |
|
| A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20240125 |
|
| R150 | Certificate of patent or registration of utility model |
Ref document number: 7430944 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R150 |